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WO2024018037A1 - Microalgues exprimant glp-2 et leurs utilisations - Google Patents

Microalgues exprimant glp-2 et leurs utilisations Download PDF

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Publication number
WO2024018037A1
WO2024018037A1 PCT/EP2023/070232 EP2023070232W WO2024018037A1 WO 2024018037 A1 WO2024018037 A1 WO 2024018037A1 EP 2023070232 W EP2023070232 W EP 2023070232W WO 2024018037 A1 WO2024018037 A1 WO 2024018037A1
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seq
host cell
disorders
treatment
biomass
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Inventor
Raffaele Ingenito
Marco MATTU
Giuseppe Martelli
Rosa Paola RADICE
Maria Carmela PADULA
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Bioinnova SRLS
Naturamla Srl
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Bioinnova SRLS
Naturamla Srl
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/575—Hormones
    • C07K14/605—Glucagons
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C12N1/12—Unicellular algae; Culture media therefor
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/62—DNA sequences coding for fusion proteins
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Definitions

  • the present invention relates to microalgae capable of expressing glucagon-like peptide (GLP), preferably GLP-2 or analogs of GLP-2, and uses thereof in pharmaceuticals or as a dietary supplement in the treatment and prevention of disorders affecting the gastrointestinal system, including in particular acute and chronic inflammatory diseases.
  • GLP glucagon-like peptide
  • a bioactive compound is any compound present in foods, animals, or plants that has an effect on the body that consumes it.
  • bioactive ingredients are those that, when inserted in foods, provide some effect of improvement to health.
  • Bioactive ingredients or active ingredients often referred to as functional ingredients, are compounds extracted from a source food, such as fruits, cereals, vegetables, and food processing residues, which preserve their characteristics even after extraction.
  • nutraceutical product or “functional food” defines all those foods, fresh or processed, which, through their normal consumption within the normal diet, by containing bioactive compounds, offer potential improvement in human health by reducing the occurrence of certain diseases.
  • These functional foods aim to promote and support the well-being of the human body, and their development is extremely valuable from the perspective not only of treatment, for patients with gastointestinal (GI) disorders, but also of complementary and alternative prevention and containment of these diseases.
  • GI gastointestinal
  • Typical functional foods contain, within them, specific natural or additional minerals, vitamins, fatty acids, dietary fiber and/or biologically active substances such as, for example, phytochemicals, antioxidants, probiotics and, also, proteins.
  • GI diseases and, in particular, chronic inflammatory bowel diseases (IBDs) such as inflammatory bowel syndrome, Crohn's disease, and ulcerative colitis are characterized by a chronic inflammatory state and, in recent years, their incidence, and prevalence has increased considerably in synergy with their severity.
  • IBDs chronic inflammatory bowel diseases
  • triggers for such intestinal dysfunctions that can result in severe intestinal failure include all conditions for which there is a change in the intestinal microbiota (drug use, alcohol, presence of malignant neoplasms, etc.).
  • IBDs Irritable bowel syndrome
  • GLP glucagon-like peptide
  • GLP-2 reversed the atrophy of the intestinal mucosa induced by parenteral nutrition and accelerated the process of endogenous adaptation in rats that had a large part of the small intestine severed.
  • GLP-2 also significantly attenuated intestinal damage, and weight loss, in rat models with indomethacin-induced enteritis.
  • GLP-2 also exerted reparative and cytoprotective actions in rodents in which intestinal inflammation was induced by chemotherapy treatments.
  • Human clinical results at the same time, have fully elucidated the beneficial effects of GLP-2 in patients with intestinal mucosal damage, confirming its ability to significantly increase energy uptake, reduce water loss and nitrogen absorption in treated subjects compared with controls.
  • Body weight and lean body mass moreover, increased, as opposed to fat mass, which, on the other hand, decreased; the time required for gastric emptying was also increased by 50%.
  • histological analysis of intestinal tissue revealed how the depth and height of intestinal villi also increased following GLP-2 administration (Julie Lovshin, Daniel J. Drucker, in Encyclopedia of Endocrine Diseases, 2004).
  • GLP-2 is a peptide hormone composed of 33 amino acids released by intestinal L-endocrine cells upon nutrient ingestion and exerts its trophic action on the epithelium of the small and large intestine through stimulation of cell proliferation and inhibition of apoptosis. GLP-2 also upregulates intestinal glucose transporter activity and reduces gastric emptying and gastric acid secretion. GLP- 2 activity is regulated, in part, through renal clearance and cleavage by dipeptidyl peptidase IV aminopeptidase. The actions of GLP-2, in addition, are transduced by the glucagon-like peptide-2 receptor (GLP-2R), which represents a member of the G-protein-coupled receptor superfamily. GLP- 2R is involved in the increased adenylate cyclase activity.
  • GLP-2R glucagon-like peptide-2 receptor
  • GLP-2 is obtained primarily by chemical synthesis or biologically by recombinant DNA (cDNA) technology, with several purification steps downstream in the process. Based on all these considerations, it is evident that there is a need to make available systems capable of expressing a bioavailable, low-cost form of GLP-2 for use in the treatment and prevention of GI disorders that is easily obtainable and suitable for preferably oral administration.
  • Microalgae are single-celled plant organisms belonging to both the prokaryotic and eukaryotic kingdoms. Microalgae produce energy through the photosynthetic process, removing carbon dioxide from the atmosphere and releasing oxygen at the end of the process.
  • microalgae Their special position in phylogenetic evolution makes them model organisms, as they share characteristics common to both bacteria and higher plants.
  • the unicellular nature of microalgae means that they must respond to environmental stresses quickly and efficiently (to ensure their survival), maintaining the genome in a highly reactive euchromatic state.
  • Number of studies, concerning microalgae, derive from the large production of secondary metabolites that are perfectly placed in the nutraceutical and pharmaceutical fields, these being potent antioxidants, essential amino acids and polyunsaturated fatty acids (co3 and co6).
  • Microalgae comprise a wide range of mainly aquatic, unicellular, and eukaryotic organisms (including green algae, diatoms, and brown algae) that engage in photosynthetic activity, and contain cytoplasmic organelles such as mitochondria and chloroplasts.
  • the chromatin structure of microalgae is distinct from other eukaryotic organisms; in fact, this is heavily colored highlighting a more compact nucleosomal structure and a close association of DNA with histone protein components.
  • Microalgae are considered an important source of healthy nutrients for human needs and are important in that biomass and biofuels can also be derived from their cultivation. Genetic engineering and stable expression (maintained over multiple generations) of different transgenes would open new horizons and greatly enhance the value and opportunity of cultivating microalgae for purposes of improving healthy well-being. However, as described earlier, it has been difficult to achieve a stable and sufficiently high level of gene expression, so a new methodology to help overcome this obstacle is extremely useful. Such an approach must consider gene silencing related to the unique and resistant histone presence of microalgae, including green microalgae.
  • the present invention reports a totally innovative approach regarding the use of specific microalgal strains and specific growth and reproduction techniques to express and produce GLP-2 and/or GLP- 2 peptide analogs as an endogenous metabolite to be used, for example in the form of freeze-dried biomass or humid biomass absorbed over dried functionalized food flour, as a bio-active material for animal and human use.
  • nucleic acid molecule comprising or consisting of: a. a coding polynucleotide sequence having at least 85% homology to SEQ ID No. 28 or which matches perfectly or is complementary to SEQ ID No. 28 or having at least 85% homology to any of SEQ ID No. 53-64 or which matches perfectly or is complementary to any of SEQ ID No. 53-64; and b. at least one linker polynucleotide sequence with at least 85% homology to any of SEQ ID No 1-21; wherein the linker polynucleotide sequence is linked to the 5 'end and/or the 3' end of the coding polynucleotide sequence.
  • said nucleic acid molecule comprises or consists of: c. a coding polynucleotide sequence having at least 85% homology to SEQ ID No. 28 or which matches perfectly or is complementary to SEQ ID No. 28 or which matches perfectly or is complementary to SEQ ID No. 28 or having at least 85% homology to any of SEQ ID No. 53-64 or which matches perfectly or is complementary to any of SEQ ID No. 53-64; and d. a linker polynucleotide sequence with at least 85% homology to any of SEQ ID No 1- 21, linked to the 5' end of the coding polynucleotide sequence; and e. a linker polynucleotide sequence with at least 85% homology to any of SEQ ID No 1- 21, linked to the 3' end of the coding polynucleotide sequence.
  • said nucleic acid molecule has at least 85% homology to any of SEQ ID No 29-42 or has at least 85% homology to any of SEQ ID No 65-76.
  • an host cell comprising said nucleic acid molecule, wherein said host cell is an algal cell; preferably said host cell belongs to the class of Chlorophyceae and/or to the class of Trebouxiophyceae; still preferably said host cell to the species Haematococcus spp. and/or Chlorella spp.; even more preferably said host cell belongs to Haematococcus pluvialis and/or Chlorella vulgaris.
  • the invention provides said host cell for use in the treatment and/or prevention of intestinal dysbiosis, chronic inflammatory bowel disease (IBD), for the treatment of irritable bowel disorders, for the containment of the progression of osteoporosis, for the improvement of disorders resulting from bone resorption, to strengthen and improve the intestinal microbiota in human or veterinary animals; preferably said host cell according is for use in the treatment and/or prevention of inflammatory bowel syndrome, Crohn's disease and ulcerative colitis and/or intestinal dysbiosis due to eating disorders and autism spectrum disorders.
  • IBD chronic inflammatory bowel disease
  • GLP2 expressing algal biomass comprising at least one host cell as defined above and/or a lysate and/or an extract of said host cell.
  • said biomass comprises the GLP2 peptide of SEQ ID No. 43 and/or GLP2 peptide analogs comprising SEQ ID No 43 or comprises the GLP2 peptides of SEQ ID No. 44-52 and/or analogs comprising SEQ ID No. 44-52.
  • It is a further object of the invention a method for obtaining said GLP2 expressing algal biomass comprising the following steps: a. induce thermal stress in a culture of microalgae by heating at a temperature between 35° and 50° C for a time between 300 and 600 seconds; b. expose the microalgae culture to UV rays (UV-A, UV-B and UV-C) for one or more time intervals between 5 and 15 min; c. inoculate the culture treated in step b in fresh liquid medium; d.
  • UV rays UV-A, UV-B and UV-C
  • a mixture of lytic enzymes comprising at least one of the following enzymes: cellulase, cellulase CP, hemicellulose, chitinase, 0-D- glucanase, macerozyme, helicase, driselasi, lytic enzyme L, pectinase, protease, xylanase, cutinase, P-D-glucuronidanase, cellobiohydrolase, mixtures of them; e.
  • step d incubate the microalgae culture treated in step d) at a temperature between 30 - 40° C for 4-8 h; f. combine the culture incubated from step e) in 10 ml of culture medium, with 0.1 - 0.5% of a solution comprising the isolated nucleic acid as defined in the previous paragraphs, in the presence of 10-35 % polyethylene glycol (PEG-X).
  • PEG-X polyethylene glycol
  • the microalgae preferably belong to the Chlorophyceae class and or to the Trebouxiophyceae, more preferably belong to the Haematococcus spp. and/or Chlorella spp., even more preferably belong to the Hematococcus pluvialis species and/or Chlorella vulgaris.
  • IBD chronic inflammatory bowel disease
  • composition comprising the host cell of the invention or the GLP-2 expressing algal biomass of the invention and at least one pharmacologically acceptable excipient.
  • said pharmaceutical composition is for use in the treatment and/or prevention of intestinal dysbiosis and inflammatory bowel disease (IBD), for the treatment of disorders deriving from irritable bowel, for the containment of the progression of osteoporosis, for the improvement of disorders resulting from bone resorption, to strengthen and improve the intestinal microbiota in human or veterinary animals; preferably for use in the treatment and/or prevention of inflammatory bowel syndrome, Crohn's disease and ulcerative colitis and/or intestinal dysbiosis due to eating disorders and autism spectrum disorders.
  • IBD intestinal dysbiosis and inflammatory bowel disease
  • IBD chronic inflammatory bowel diseases
  • FIG. 1 Effect of natural products with pharmacological properties, extracted from engineered algae, in a mouse model of 2,4,6-trinitrobenzene sulfonic acid (TNBS)-induced colitis.
  • A Effect of tested compounds on body weight in the TNBS-induced colitis mouse model. Body weight was monitored daily until sacrifice. Weight change over time is expressed as a percentage of initial body weight. Lines indicate the mean ⁇ standard error of each experimental group;
  • B Effect of derivatives extracted from artificial seaweed on colonic length in TNBS-induced colitis mice. At the end of the study (day +3 from TNBS infusion), the two spots were excised and the length was determined with a slide caliper. Data shown represent mean + ES of 8 mice/group.
  • FIG. 1 Cell viability reported as a function of the tested dose of algae, expressed as the absorbance value of formazan crystals that is directly proportional to cell viability.
  • A 24 hours;
  • B 48 hours;
  • FIG. 1 Schematic representation showing the method of the invention according to one embodiment.
  • Chlorophyceae are one of the classes of green microalgae, distinguished mainly on the basis of ultrastructural morphology. Usually their coloration is caused by the predominance of photosynthetic pigments such as chlorophyll a and chlorophyll b.
  • the chloroplast can have a discoidal, plate-shaped, reticulate, cup-shaped, spiral or ribbon-like conformation depending on the species to which it belongs.
  • pyrenoids located within the chloroplast. Pyrenoids, in turn, contend with various proteins including starch.
  • Some species of green microalgae can store nutrient sources in the form of oily droplets. They usually have a cell wall consisting of an inner layer of cellulose and an outer layer of pectose.
  • Trebouxiophyceae is a further class of green microalgae suitable for the purposes of the invention, in particular Chlorella spp.
  • the present invention is related to the production of exogenous proteins within a host cell, represented by a microalgal cell belonging to the class Chlorophyceae and/or Trebouxiophyceae.
  • the host cell is used as a biofactory for protein production.
  • the recombinant Chlorophyceae host cell is Haematococcus spp.
  • the recombinant Trebouxiophyceae host cell is Chlorella spp.
  • the host cell is Haematococcus pluvialis and/or Chlorella vulgaris.
  • nucleic acid molecule comprising or consisting of: a. a polynucleotide coding sequence having at least about 50 percent homology, preferably at least about 60 percent, preferably at least about 70 percent, preferably at least about 85 percent, at least about 90 percent, at least about 95 percent, at least about 98 percent to SEQ ID No. 28 or that appears perfectly or is substantially complementary to SEQ ID No. 28, or its fragments or functional derivatives; b.
  • linker polynucleotide sequence with at least about 50 percent homology, preferably at least about 60 percent, preferably at least about 70 percent, preferably at least about 85 percent , at least about 90 percent, at least about 95 percent, at least about 98 percent to any one of SEQ ID No 1-21; wherein the linker polynucleotide sequence is operatively bound to the 5' and/or 3' end of the coding sequence.
  • homology refers to sequences characterized by similarity at the nucleotide level or amino acid level as discussed herein.
  • a nucleic acid molecule that is complementary to the nucleotide sequence of SEQ ID NO 28 is one that is sufficiently complementary to the nucleotide sequence of SEQ ID NO 28 that it can hydrogen bond with few or no mismatches to the nucleotide sequence shown in SEQ ID NO 28 thereby forming a stable duplex.
  • binding means the physical or chemical interaction between two polypeptides or compounds or associated polypeptides or compounds or combinations thereof. Binding includes ionic, non-ionic, van der Waals, hydrophobic interactions, and the like.
  • a physical interaction can be either direct or indirect. Indirect interactions may be through or due to the effects of another polypeptide or compound. Direct binding refers to interactions that do not take place through, or due to, the effect of another polypeptide or compound, but instead are without other substantial chemical intermediates.
  • the linker polynucleotide sequences with at least about 50 percent homology, preferably at least about 60 percent, preferably at least about 70 percent, preferably at least about 85 percent, at least about 90 percent, at least about 95 percent, at least about 98 percent to any one of SEQ ID No 1-21 are two and are bound at the 5' and 3' ends, respectively, of the coding polynucleotide sequence.
  • the isolated nucleic acid molecule is of sequence corresponding to SEQ IDs No 29-42, or functional fragments and derivatives.
  • a host cell comprising the isolated nucleic acid molecule as defined above, wherein said host cell is an algal cell, preferably said host cell belongs to the class Chlorophyceae and/or Trebouxiophyceae, preferably said host cell belongs to the species Haematococcus spp. and/or Chlorella spp..
  • the invention also relates to the host cell for use in the treatment and/or prevention of intestinal dysbiosis, chronic inflammatory bowel disease (IBD), for the treatment of disorders resulting from irritable bowel syndrome, for the containment of the progression of osteoporosis, for the improvement of disorders resulting from bone resorption, for strengthening and improving the intestinal microbiota, preferably for use in the treatment and/or prevention of inflammatory bowel syndrome, Crohn's disease and ulcerative colitis and/or intestinal dysbiosis due to eating disordersand autism spectrum disorders.
  • IBD chronic inflammatory bowel disease
  • IBD chronic inflammatory bowel disease
  • osteoporosis for the improvement of disorders resulting from bone resorption
  • strengthening and improving the intestinal microbiota preferably for use in the treatment and/or prevention of inflammatory bowel syndrome, Crohn's disease and ulcerative colitis and/or intestinal dysbiosis due to eating disordersand autism spectrum disorders.
  • the invention is directed to an algal biomass comprising at least one host cell as defined above and/or a lysate and/or extract and/or secretion of said host cell and comprising expressed GLP2 (SEQ ID No 43) and GLP2 peptide analogs .
  • a GLP2 peptide analog is a peptide comprising the GLP2 sequence HADGSFSDEMNTILDNLAARDFINWLIQTKITD (SEQ ID No. 43) elongated at the N or C terminal with additional aminoacids corresponding to or encoded by the specific linkers of the invention.
  • Said GLP2 analog is a functional analog as it maintains the same biological properties of the GLP2 peptide of SEQ ID No 43.
  • an object of the invention is a method for obtaining an algal biomass expressing GLP2 and/or GLP2 peptide analogs comprising the following steps: a. Induce heat stress in a culture of microalgae belonging to the class Chlorophyceae by heating at a temperature between 35 and 50°C for between 300 and 600 seconds; b. Expose the microalgae culture to UV light (UV-A, UV-B and UV-C) for one or more time intervals between 5 and 15 min; c. Inoculate the culture treated in step b into fresh liquid medium to restore normal biophysiological conditions; d.
  • UV light UV-A, UV-B and UV-C
  • a macerating solution consisting of 16-50% (v/v) 0.35 M mannitol and 0.2-0.6% lytic enzyme mixture comprising at least one of the following enzymes: cellulase, cellulase CP, hemicellulase, chitinase, 0-D-glucanase, macerozyme, helicase, driselase, lytic enzyme L, pectinase, protease, xylanase, cutinase, P-D-glucuronidanase, cellobiohydrolase, or mixtures thereof; e.
  • step (d) Incubate the microalgae culture treated in step (d) at a temperature of 30 to 40°C for 4 to 8 h; f. combine the culture incubated from step (e) in 10 ml of culture medium, with 0.1 to 0.5 % of a solution comprising the nucleic acid isolated according to any of claims 1 or 2, in the presence of 10 to 35 % polyethylene glycol (PEG-X).
  • PEG-X polyethylene glycol
  • microalgae belong to the classes Chlorophyceae, Trebouxiophyceae, preferably microalgae belong to the species Haematococcus spp., Chlorella spp. Further forming an object of the invention is an algal biomass expressing GLP-2 and /or GLP-2 peptide analogs obtainable by the method defined above.
  • the present invention also relates to a pharmaceutical composition
  • a pharmaceutical composition comprising the invention's host cell or biomass and at least one pharmacologically acceptable excipient.
  • said pharmaceutical composition is for use in the treatment and/or prevention of intestinal dysbiosis, chronic inflammatory bowel disease (IBD), for the treatment of disorders resulting from irritable bowel syndrome, for the containment of the progression of osteoporosis, for the improvement of disorders resulting from bone resorption, for strengthening and improving the intestinal microbiota, preferably for use in the treatment and/or prevention of inflammatory bowel syndrome, Crohn's disease and ulcerative colitis, and/or intestinal dysbiosis due to eating disorders and autism spectrum disorders.
  • IBD chronic inflammatory bowel disease
  • a food supplement or drinking product comprising the host cell or biomass as defined above.
  • IBD chronic inflammatory bowel disease
  • the products of the invention find use in treatment, prevention and as supplements in situations of intestinal dysbiosis preferably due to eating disorders and autism spectrum disorders.
  • Efficient growth medium is defined as any culture medium in which a microalgal cell, such as the Chlorophyceae cell, is usually grown. Such medium typically includes an aqueous phase containing assimilable sources of carbon, nitrogen and phosphate, as well as mineral salts, metals and other appropriate nutrients such as, for example, vitamins.
  • suitable media and growth conditions are described in the "Examples" section.
  • Cells of the present invention can be cultured in conventional fermentation photobioreactors, shaking flasks, test tubes, microtiter plates, and Petri dishes. Culture can be carried out at appropriate temperature, pH and oxygen content for the recombinant cell.
  • the term "transformation” identifies any methodology by which an exogenous nucleic acid molecule (i.e., a recombinant nucleic acid molecule) can be inserted within microbial cells.
  • an exogenous nucleic acid molecule i.e., a recombinant nucleic acid molecule
  • transformation is used primarilyto describe a genetic, heritable change caused by the acquisition of exogenous nucleic acids by the microorganism, and is essentially synonymous with the term “transfection.”
  • Several methodologies suitable for the introduction of exogenous nucleic acid molecules inside algal hostcells are known, such as (i) shotgun with gold particles, (ii) electroporation, (iii) micro injection, (iv) lipofection, (v) adsorption, (vi) infection, and (vii) protoplastic fusion.
  • a biomass according to the present invention is a composition comprising transformed algal cells and/or extracts and/or lysates or other derivatives. More specifically, the biomass of the invention is obtained after lysis of the cell culture and comprises, inter aha, the GLP2 peptide and the GLP2 peptide analogs.
  • a methodology for transforming a competent algal host cell involves two main steps: a) pretreating the host cell with an enzyme, and b) introducing an exogenous nucleic acid molecule into the host cell.
  • the method of the invention is well explained in Figure 4.
  • the enzyme can have cellulase, protease, P-glucoronase and various combinations of these activities.
  • the expression system of the invention which allows the GLP-2 protein to be expressed within the algal cell, includes regulatory control elements that are active in microalgal cells. Many of these elements, including several promoters, are active in different species; therefore, the novel regulatory sequences, described as aspects of the invention, can be used not only in the algal cells described here, but also in cells belonging to different species characterized by the same evolutionary mechanisms .
  • the design and construction of theexpression systems covered by the invention use standard biomolecular technologies known to persons skilled in the art. See, for example, Sambrook et al., 2001, Molecular Cloning: A Laboratory Manual, 3rd edition.
  • the expression system or expression vector comprises a polynucleotide sequence encoding for the GLP-2 protein associated with any promoter sequence, or 5' linker, and possibly a terminator sequence, or 3' linker.
  • the 5' linker, the GLP-2 coding sequence and the 3' linker are operatively linked so that they are functional within the host cell.
  • the expression system may also include additional regulatory sequences that are functional within the genome of the host cell. Inducible or constitutively active sequences can be used. Suitable control elements, also include any regulatory element associated with the expression of the nucleicacid molecules described here.
  • the present invention is also directed to the algal host cell comprising the expression system described above and/or to a biomass obtained from or comprising said algal cell.
  • the expression system of the invention preferably comprises at least one of the nucleic acid molecules isolated in the present invention and described herein.
  • all genetic elements of the expression system are sequences associated with previously isolated nucleic acid molecules.
  • the nucleic acid sequence encoding for the GLP-2 protein, or coding sequence is stably integrated into the genome of the host cell, while in others, said coding sequence is operatively linked to a promoter linker sequence and/or a terminator linker sequence, both of which are functional in the host cell.
  • the linker sequences to which the coding sequence is operatively linked include, but are not limited to, the novel nucleic acid sequences described inthe present invention.
  • the coding sequence is optimized for the codon belonging specifically to the host cell of Haematococcus spp. and/or Chlorella spp. so as to maximize translation efficiency.
  • the recombinant algal cell that is the subject of the invention is capable of expressing therapeutic proteins.
  • a "therapeutic protein,” as used herein, includes proteins useful for the treatment or prevention of diseases, pathological conditions, and various disorders in both animals and humans.
  • prevention and treatment refer to both therapeutic treatment and prophylactic or preventive measures in which the objective is to prevent or slow (reduce) an undesirable pathophysiological condition, disease, or disorder, or to achieve beneficial or desired clinical results.
  • beneficial or desired clinical results include, butare not limited to, the alleviation of symptoms or signs associated with a pathological condition or disorder of normal physiology; reducing the severity of a condition, disease, or disorder; stabilization of a condition, disease, or disorder (or better, situations in which the condition, disease, or disorder is stable and does not worsen over time) delay in the onset or progression of the condition, disease, or disorder; improvement of the condition, disease, or disorder; remission (total or partial and detectable or undetectable) of the condition, disease, or disorder; or enhancement or improvement of a condition, disease, or disorder.
  • Treatment includes eliciting a clinically meaningful response without excessive side effects and also prolonging survival over expected survival if treatment is not received.
  • the therapeutic protein is glucagon-like peptide 2 (GLP-2) and analogs thereof.
  • GLP-2 glucagon-like peptide 2
  • Protein produced or expressed by the algae cell according to the invention can be produced on a commercial scale.
  • Commercial scale includes protein production from a microorganism grown in an aerated bioreactor (biofermentor) of size > 100 L, > 1,000 L, > 10,000 L, or > 100,000 L. In some forms of implementation, commercial-scale production is performed in an aerated biofermentor with agitation.
  • the protein produced by the algae cell can also accumulate within the cell or can be secreted by the cell, for example, into the culture medium as a soluble protein.
  • the protein produced can be recovered from the cell, the culture medium, or the fermentation medium in which the cell itself is grown.
  • the same biomass expressing the GLP-2 protein and GLP-2 analog can be used directly for the purposes of the invention, which include therapeutic and preventive uses and nontherapeutic uses, for example, in the preparation of
  • the present invention is directed to a method for producing a recombinant protein; the methodology also includes culture conditions for the microalgal cells of the invention such that a polynucleotide sequence coding for a protein can be expressed.
  • Proteins produced by the method elaborated in this invention can also be purified using a variety of standard protein purification techniques such as, but not limited to, affinity chromatography, ion exchange chromatography, filtration, electrophoresis, hydrophobic interaction chromatography, gel filtration chromatography, reversed phase chromatography, chromatographyusing concanavallin A, chromatofocusing, and differential solubilization.
  • proteins produced by the method described in the present invention are isolated in "substantially pure” form. In this context, “substantially pure” refers to a purity that allows effective use of the protein as a commercial product and/or ingredient to be used in combination with others.
  • the recombinant protein accumulates within the cell and is recovered from the cell; in some embodiments, the host cell of the method belongs to the Chlorophyceae class, while in other embodiments, the host cell is from Haematococcus spp. In some embodiments, the host cell belongs to the Trebouxiophyceae class, while in other embodiments is Chlorella spp.
  • the recombinant protein is a therapeutic protein, a food enzyme or an industrial enzyme. In some realizations, the recombinant protein is GLP-2. In some realizations, the recombinant protein is a therapeutic protein that includes a secretion signal sequence, such as the GLP2 analog.
  • Isolated nucleic acid molecules or polynucleotide sequences that constitute the expression systemin the algal cell form the object of the invention.
  • the nucleic acid sequences described here include the 5' and 3' linker sequences and coding sequences, particularly the GLP-2 coding sequence.
  • An isolated nucleic acid molecule can be a DNA molecule, RNA molecule (e.g., mRNA) or derivatives of them (e.g., cDNA).
  • nucleic acid molecule refers principally to the physical nucleic acid molecule, and although the phrases “nucleic acid sequence” or “polynucleotide sequence” refer primarily to the sequence of nucleotides present on the nucleic acid molecule, the phrases are used interchangeably, especially in reference to a nucleic acid molecule, polynucleotide sequence, or nucleic acid sequence encoding a protein.
  • a nucleic acid molecule isolated by the present invention is produced using recombinant DNA technology (such as, for example, cloning and amplification by polymerase chain reaction (PCR)) or by chemical synthesis.
  • PCR polymerase chain reaction
  • Isolated nucleic acid molecules include naturally occurring nucleic acid molecules and their homologs, including, but not limited to, naturally occurring allelic variants and modified nucleic acid molecules in which nucleotides have been inserted, deleted, substituted, and/or reversed in such a way that these modifications provide the desired effect on the sequence, function, and/or biological activity of the encoded peptide or protein.
  • a double-stranded DNA present in this invention includes a single-stranded DNA and its complementary strand, the sequence of which mirrors the sequence of the single-stranded DNA.
  • nucleic acid molecules of the present invention may be double-stranded or single- stranded and also include those nucleic acid molecules that form stable hybrids under high "stringency" conditions with a sequence of the invention and/or with a sequence complementary to a sequence of the invention. Methods for tracing a complementary sequence are known to experts in the field.
  • protein includes single-chain polypeptide molecules as well as multiple polypeptide complexes in which the individual constituent polypeptides are bound through covalent and noncovalent means.
  • polypeptide includes peptides of two or more amino acids in length, typically having more than 5, 10 or 20 amino acids.
  • the human GLP-2 peptide is a 33 amino acid peptide, having the following sequence: HADGSFSDEMNTILDNLAARDFINWLIQTKITD (SEQ ID No. 43).
  • GLP-2 peptide refers also to peptides of other mammals, including:
  • the new nucleic acid molecules of the present invention can be used in any genus of microalgae in which they are found to be functional.
  • the nucleic acid molecules are used in algae belonging to the class Chlorphyceae.
  • recombinant nucleic acid molecules are used in the species Haematococccus spp.
  • recombinant nucleic acid molecules are used in the class of Trebouxiophyceae and/or in the species Chlorella spp.
  • a recombinant microorganism has a genome that has been modified (i.e., mutated or changed) from its natural (i.e., naturally occurring or wild-type) form using recombinant technology.
  • a recombinant microorganism according to the present invention may include a microorganism in which nucleic acid molecules have been inserted, deleted, or modified (i.e., mutated, e.g., by nucleotide insertion, deletion, substitution, and/or inversion) such that the modifications provide the desired effect within the microorganism.
  • the present invention is directed to the 5' and 3' linker sequences.
  • the 5' or promoter linker is a DNA sequence that directs transcription of a coding region associated with it.
  • the 3' or terminal linker is the gene sequence that marks the end of transcription of genomic DNA.
  • the linker of the invention is any of the following sequences: CGGGGCAACTCAAGAAATTC (SEQ ID No 1) GTCTGGCCGAGGTCTGGTTCCTGTGCC (SEQ ID No 2)
  • CCGGACTGCCATAGCACAGCTAGACGA (SEQ ID No 11) GTCTGGCCGAGGTCTGGTTCCTGCCTAG (SEQ ID No 12) ACTGACTGCCATAGCACAGCTAGACGA (SEQ ID No 13) ATTTGCTGCATGACTGGATCAATGCGACGA (SEQ ID No 14) GTCTGGCCTGACGTATGATCGATGCCATAAATGC (SEQ ID No 15) ATGCCCTGATCCCAATGATGGACGA (SEQ ID No 16) GTCTGGCCGAAACTGATTTGGCCATGAC (SEQ ID No 17) GAGCGTGCTGAAATGCATGCGACGA (SEQ ID No 18) GTCTGGCCCCCGGGTATAGTAGCTGAC (SEQ ID No 19) CCCGGGTATAGTAGCTGACTGCGACGA (SEQ ID No 20) GTCTGGGAGCGTGCTGAAATGCATG (SEQ ID No 21)
  • nucleic acid molecule comprising a polynucleotide sequence that is at least 50%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to any one of SEQ ID NO: 1 - SEQ ID NO:21, in which the polynucleotidesequence functions as a promoter and/or terminal linker at least in algae belonging to the class Chlorophyceae.
  • the invention also relates to an isolated nucleic acid molecule comprising a polynucleotide sequence that hybridizes with any of the SEQ ID NO:1 - SEQ ID NO:21 sequences or that hybridizes with a polynucleotide sequence that is at least 95 percent identical to any of the SEQ ID NO: 1 - SEQ ID NO:21 sequences.
  • the isolated nucleic acid molecule may include a polynucleotide sequence that is completely complementary to any of the SEQ ID NO: 1 - SEQ ID NO:21 sequences or to a polynucleotide sequence that is at least 95% identical to any of the SEQID NO:1 - SEQ ID NO:21 sequences.
  • Microalgae belonging to the genus Chlorophyceae, were grown in the culture medium shown in Table 1, under illumination with an intensity of 120 mmol photons m s -2-1 in an alternating cycle 25 of 16 h of light and 8 h of dark, at a temperature of 25 °C.
  • the cultures were agitated by mechanical shaker (g24 environmental incubator shaker, American Laboratory Trading) at 70 rpm throughoutthe growth time.
  • Table 1 composition of the growth medium
  • the percentages shown in the table may vary depending on the species, genotype and initial concentration of microalgae cultures.
  • microalgae-specific genes related to the photosynthesis pathway were analyzed.
  • the following sequences were derived from specific algal sequences:
  • CACATGCCATCCGAGTCGTC (SEQ ID No 7)
  • ATGGCCACGC SEQ ID No 9
  • CTCTACCCAC (SEQ ID No 10)
  • the sequence of the human GLP-2 gene is as follows:
  • oligonucleotides comprising the human GLP2 gene sequence and at least one promoter linker, preferably a promoter linker and a terminal linker (underlined in the list below), derived from the specific algal sequences and previously described, were then synthesized:
  • the sequences of the GLP-2 gene of other mammals are as follows:
  • oligonucleotides comprising the GLP2 gene sequence from other species and at least one promoter linker, preferably a promoter linker and a terminal linker (underlined in the list below), derived from the specific algal sequences and previously described, were also synthesized by using the same method of the invention:
  • the microalgae culture was resuspended at a ratio of 1 :5 to 1 :25, depending on cell concentrations, in a macerating solution which constitutes a lytic mixture.
  • the macerating solution contained 16- 50% (v/v) 0.35 M mannitol and 0.2-0.6% lytic enzyme mixture (Table 2).
  • the lytic mixtures were then incubated at 30-37°C for 4-8 h.
  • the composition of the lytic mixture, temperature and incubation time changed according to the microalgae species.
  • microalgae solutions after treatment with the lytic mixture were combined, in a final volume of 10 ml of culture medium, with 0.1 to 0.5 % v/v of polynucleotide diluted 2, 5, 10, 20, 25, or 50 times (initial concentration 100 pM), depending on the nucleotide length and the resultingmolecular weight of each polynucleotide (Table 3)
  • Steps i, ii and iii are repeated 2 to 5 times.
  • the pellet is resuspended in 1 to 50 ml of standard culture medium. Centrifugation speed and duration vary according to the concentrations of PEG used (Table 4)
  • Pri GLP Pri GLP regl - Forward AGACATGCTGATGGTTCTTT (SEQ ID No 22) Pri GLP reg2 - Forward TCTCTGATGAGATGAACACC (SEQ ID No 23) Pri GLP reg4 - Forward GATTTCCCAGAAGAGGTCG (SEQ ID No 24) Pri GLP regl - Reverse AACATTTCAAACATCCCACG (SEQ ID No 25) Pri GLP reg2 - Reverse GCAGGTGATGTTGTGAAGAT (SEQ ID No 26)
  • the amplification protocol is as suggested in the Phire Plant Direct PCR Master Mix brochure. (Thermo ScientificTM - Cat. Number: Fl 60S - https : //www. thermofisher , com/ order/ catalog/product/F 160S )
  • Mass Spec analysis was used to determine the concentration of the GLP-2 and GLP-2 analogs in dried biomass by lyophilization.
  • the humid microalgae biomass expressing GLP-2 was collected and freeze-dried to obtain a green powder and 100 mg of this material used to extracted the desired peptide hormone.
  • Ipl of supernatant were analysed by using a AB-sciex 5500 QTRAP® system with a HPLC chromatography system Exion LCTM.
  • the mobile phase was generated by mixing eluent A (0.1 % Eormic Acid in water) and eluent B (0.1 % Eormic Acid in acetonitrile) and the flow rate was 0.200 mL/min.
  • Chromatographic gradient was from 20% to 90% in 4 min, hold for 2 min, then return to 20 % in 1 min. Tandem mass spectrometry was performed using Turbo VTM ion source operated in positive ion mode, and the multiple reaction monitoring (MRM) mode was used for the selected analytes.
  • MRM multiple reaction monitoring
  • GLP2 human glucagon-like peptide 2
  • a human glucagon-like peptide 2 (GLP2) ELISA kit (0.156-10 ng/mL) was used. 100 mg of dry algal biomass, obtained by freeze- drying the crude product, was dissolved in 10 mL of water containing 50% acetonitrile and 0.1% trifluoroacetic acid. The extraction step performed is reported in the materials and methods, in fact, the solution was sonicated for 15 min and then, by centrifugation, the algal biomass was separatedfrom the supernatant. The supernatant was lyophilized and 100 pl was resuspended in diluent buffer (provided by the kit).
  • diluent buffer provided by the kit.
  • the analysis protocol used includes the following steps:
  • the amount of peptide present in the sample is in the range of 150 to 2000 pg/ml.
  • IEC-6 cells 5x10 3 IEC-6 cells were seeded and allowed to grow overnight.
  • CTRL and GLP-2 cells of Haematococcus spp were sonicated and filtered to separate the precipitated phase from the liquid phase.
  • Different concentrations of algal extracts were tested (starting from 25% v/v. Serial 1:2 dilutions were made until the final concentration of 0.39 % v/v was reached).
  • MTT assay was conducted after 24 and 48 hours to check cell viability. The protocol used involves the following steps:
  • mice Two different efficacy studies were conducted in animal models (mouse) with the aim of verifying non toxicity in vivo and measuring the efficacy of GLP2 produced in alga .
  • IBD Inflammatory bowel disease
  • TNBS 2,4,6-trinitrobenzene sulfonic acid
  • GLP-2 2,4,6-trinitrobenzene sulfonic acid
  • ethanol and TNBS trinitrobenzenesulfonic acid
  • Ethanol is used as a means to effectively destroy the intestinal barrier and allow TNBS to interact with proteins in colon tissue.
  • mice After colitis induction, mice develop several manifestations of acute colitis. These include soft stool formation and occult or even bloody diarrhea. Intracolonic administration of TNBS/ethanol,in mice induces severe disease characterized by bloody diarrhea and dramatic body weight loss during the first week.
  • colitis was induced by rectal administration of 2 mg/100 ml TNBS (Sigma- Aldrich, St. Louis, MO, USA) in 45% ethanol (Merck, Darmstadt, Germany) using a vinylcatheter placed 3.5 cm proximal to the anus. During the procedure, mice were anesthetized using Rumpun/Zoletil. After instillation of the catheter, the animals were kept upright for 30 sec.
  • mice underwent identical procedures but were instilled with 45% ethanol dissolved in phosphate- buffered saline (PBS). Mice were monitored daily for survival, body weight, rectal bleeding and stool consistency. All animals were sacrificed on day 5 of the experiment for cervical dislocation.
  • PBS phosphate- buffered saline
  • mice were kept fasting for 24 h before colitis induction. At day 0, mice were anesthetized by inhalation of isoflurane, 2 mg of TNBS in 100 pL of 50% ethanol solution were administered slowly into the colon through a medical-grade catheter (3.5 F) inserted gently about 4 cm into the anus.
  • a medical-grade catheter 3.5 F
  • mice Food and water were administered ad libitum after TNBS instillation. Mice were divided into the following experimental groups (8 mice/group):
  • GLP2 algae are GLP2-expressing algae according to the invention
  • CTRL algae are control algae
  • Mesalazine an anti-inflammatory agent used in the treatment of inflammatory bowel disease (positive disease control) was administered orally by gastric gavage from three hours before the TNBS infusion and for the next three days (once daily in the morning, 4 total doses). Algae were administered orally by gavage from seven days before TNBS infusion and for the next three days (once daily in the morning, 10 total doses). Mice were sacrificed by CO asphyxiation2 in the afternoon 3 days after intrarectal administration of TNBS.
  • Blood samples were collected and frozen from 3 mice of groups 1, 3, 5 and 6. the two points were explanted, measured in length with a slide caliper, immediately frozen inliquid nitrogen, and stored at -80°.
  • PBS 1 mL/mouse
  • TNBS 100 pL in 50% ethanol/mouse solution.
  • Mesalazine 1 mL/mouse GLP2 and CTRL algae: 300pL and lOOpL/mouse (two doses for both types of algae)
  • TNBS Frequency of administration
  • Mesalazine daily from three hours before TNBS infusion until the mice are sacrificed.
  • GLP2 and CTRL algae daily from seven days before TNBS infusion until mice are sacrificed.
  • mice that were instilled with TNBS disease control developed severe disease characterized by a body weight loss of about 14% from their initial body weight.
  • Figure 1(B) shows that, macroscopically, the colon is shorter in the groups of mice treated with TNBS and both doses of CTL alga, with a reduction of about 70% compared with the positive control group (Mesalazine).
  • the DAI score is a marker of disease activity and was determined based on the methods of Murano et al. (Murano M. et al. (2000): Therapeutic effect of intracolonically administered nuclear factor K B (p65) antisense oligonucleotide on mouse dextran sulphate sodium (DSS)-induced colitis.
  • Res65 nuclear factor K B
  • DSS mouse dextran sulphate sodium
  • the health quality of mice is a marker extrapolated from the human endpoint scoring system, according to which, based on predetermined physiological or behavioral signs, a score can be assigned to the health status of the animal enrolled in the experimental study.
  • colon damage index scores colon features in TNBS-treated mice such as hyperemia, thickening and ulceration.
  • mice that received GLP2 algae had a lower mortality rate (and thus higher survival) than both the control and Mesalazine groups.
  • ASD Autism
  • mice of the BTBR T+ Itpr3tf/J strain Animal models currently used by the scientific community as a model of the autism spectrum include mice of the BTBR T+ Itpr3tf/J strain (BTBR; Meyza,K.Z., Blanchard, D.C., The BTBR mouse model of idiopathic autism. Current view on mechanisms. Neurosci. Biobehav. Rev. (2017)). In fact, presenting the 3 key symptoms (problems with socialization, communication, and repetitive behaviors), he summarizes the main biochemical, neuropathological, and behavioral features found in human pathology (13, 14). In addition, recent studies have shown that a nutritional approach can induce metabolic and behavioral changes in these mice. For example, when subjected to fatty diet from the time of weaning, such strain shows even more antisocial behavior and cognitive rigidity. This strain therefore constitutes an important investigative tool for the study of metabolic disorders related to autism (15-17).
  • Mitochondria in addition to being the main cellular energy powerhouse (contributing to the production of about 90 percent of the energy used by our body), are known to synthesize key molecules during inflammatory and oxidative processes, serving as the main source of free radicals. Therefore, it is not surprising that mitochondrial dysfunction is associated with inflammation and other metabolic disorders in which cellular oxidative damage is caused by reactive oxygen species (ROS) production that exceeds physiological antioxidant activity (20). So, mitochondrial dysfunction can be both the cause and the consequence of inflammatory processes and cause metabolic adaptations that could be protective or become progressively harmful (21).
  • ROS reactive oxygen species
  • the animals were fed a standard laboratory diet (15.88 kJ/g) for 3 weeks and were divided into two groups: the first group (control) received daily intragastric administration of vehicle (water), the second group (treated) received daily intragastric administration of Haematococcus spp- GLP2 extract of lOOpl/day for 2 weeks and 200pl/dayfor an additional 7 days (Haematococcus pluvialis was used for this experiment). Body weight and food intake were monitored daily.
  • the conceptual idea behind the invention is based on the use of microalgae as natural and green bioreactors, capable of producing and transporting molecules of interest.
  • GLP-2 as described above, plays a key role in intestinal well-being, and microalgae represent optimal carriers.
  • the platform aims to design edible microalgae to make naturally produced GLP-2 to be used directly as a carrier for gut delivery.
  • the innovation of this technology lies in the combination of an already useful product for humanhealth (microalgae high in antioxidants) with a molecule of pharmaceutical interest (GLP-2 and/or GLP-2 peptide analogs).
  • the methodology used exploits the possible ability of microalgae to incorporate exogenous DNA.
  • a polynucleotide containing the GLP-2 coding sequence was synthesized and two linkers were joined at the terminal end, coding for a structural gene of the microalgae, to enable targeted insertion.
  • No physical or biological vector was used.
  • a novel methodology was applied and special conditions were optimized that allowed the microalgae to incorporate the synthetic polynucleotide.
  • the microalgae were first selected on solid medium and at a later stage were inoculated into the specific liquid culture medium and allowed to grow following standard growth protocols.
  • the biomass was harvested and processed to obtain the extract in accordance with what was described in the experimental part.
  • the TNBS-induced colitis model has proven very useful in understanding the pathophysiology of intestinal inflammation and is a powerful tool for evaluating interventions to prevent or amelioratethe disease.
  • the in vivo study carried out in the present invention aims to evaluate the protective and antiinflammatory effect of derivatives extracted from engineered algae in an experimental mouse model of TNBS-induced colonic inflammation.
  • the hypothesis based on suggestions from the literature, is that they might have a marked effect of reconstructing the intestinal epithelium, thus reducing the process of dysbiosis resulting in anti-inflammatory activity and improvement of the intestinal microbiota. It also might be able to prevent oxidative damage by scavenging free radicals.
  • the experimental data obtained in the invention confirmed the high protective and antiinflammatory effects with improvement in clinical manifestations of colitis obtained by administering the GLP2 alga of the invention at both doses used, but especially at the lowest dose (lOOul) .
  • the in vivo model correlating with autism spectrum also confirmed that the tested product is not toxic in mice (that although genetically modified for the BTBR gene have a normal life span) and provided a clear evidence that the improvement of the epithelium quality results in less inflammation and, consequently, better quality of the microbiota. Through the gut-brais axis these effects have a positive impact on autistic behavior.
  • Mitochondrial dysfunction in autism spectrum disorders a systematic review and meta-analysis. Mol Psychiatry 2012;17:290-314. Chan DC. Mitochondria: dynamic organelles in disease, aging, and development. Cell 2006;125:1241-1252. Currais A, Goldberg J, Farrokhi C, Chang M, Prior M, Dargusch R, Daugherty D, et al. A comprehensive multiomics approach toward understanding the relationship between aging and dementia. Aging (Albany NY) 2015;7:937-955.

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Abstract

La présente invention concerne un produit de microalgues capable d'exprimer un peptide de type glucagon (GLP), de préférence GLP-2, et son utilisation dans le domaine pharmaceutique ou nutraceutique dans le traitement et la prévention de troubles affectant le système gastro-intestinal, y compris notamment des maladies inflammatoires aiguës et chroniques.
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